Human perforin permeabilizing activity, but not binding to lipid membranes, is affected by pH

The various steps that perform (PFN) a critical mediator of Innate Immune response undertakes to form a transmembrane pore remains poorly understood We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation The membrane association of PFN was calcium dependent irrespective of pH However PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5 5 even though the monomers bind to the membranes in the presence of calcium It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7 4) These results were Independently confirmed on GUV and Jurkat cells The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344 Pf-344 maps to a linear epitope within region 373-388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation the site recognized by Pf-80 is only accessible at acidic but not neutral pH Thus the Pf-80 mAb likely Interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer Into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology (C) 2010 Elsevier Ltd All rights reserved